We characterized the regulation of proline oxidase by making a POX promoter-luciferase reporter construct to test the functional role of various transcriptional factors. Among the transcriptional factors tested, PPARgamma was the most potent. Additionally, pharmacologic ligands of PPARgamma, i.e. the thiazolidinediones, commonly used drugs for type 2 diabetes, further increased the induction of POX. We showed that PPARgamma bound to its response element in the POX promoter using electrophoretic mobility shift analysis and chromatin immuno-precipitation assays. Troglitazone, a potent thiazolidinedione, induced POX by PPARgamma-dependent and -independent mechanisms. The latter is mediated indirectly through p53. The coupling of POX to PPARgamma strongly suggests that POX is involved in regulation of bioenergetics and responses to nutrient stress, a finding which led us to consider the mTOR-AMPK signaling pathway. This pathway integrates signals from growth factors, nutrients, energy levels and cellular stress to regulate protein translation and cell growth. Mutations in this pathway have been associated with a number of neoplastic phenotypes. We tested the effects of Rapamycin, an inhibitor of mTOR, LY 294002, an inhibitor of PI3-K/Akt, and 5-amino-4-carboxamide ribofuranoside (AICAR), a purine analog which activates AMP-directed protein kinase (AMPK). We found that these agents which block mTOR signaling at 3 different sites, all markedly activated POX activity. Additionally, Rapamycin by blocking mTOR inhibited protein translation and cell growth and concomitantly increased cellular ATP levels presumably to sustain a vegetative survival state. Interestingly, blockade of POX expression by POX siRNA or inhibiting POX catalytic activity with dehydroproline markedly inhibited the Rapamycin induced increase in cellular ATP. These studies suggest that proline can function as a stress substrate under the regulation of PPARgamma and the mTOR/AMPK signaling pathways. Although we showed that POX expression generated ATP under conditions of nutrient stress, the biochemical source for the ATP required elucidation. Glucose is the main source for ATP in cultured cells; therefore we tested whether glycolysis was increased by POX overexpression. Surprisingly, glycolysis measured by the conversion of (5)-3H-glucose to 3H2O was not changed with POX overexpression. In contrast, the pentose phosphate shunt (PPS) was increased more than 5-fold when POX was induced. Furthermore, with limiting glucose (.05 mM), ATP levels progressively fell. However, when POX was induced, ATP levels were maintained in the presence or absence of added proline. Presumably, the cycling of endogenous proline could mediate the effect. These findings suggest that when glucose is limiting, POX promotes the metabolism of glucose through the PPS and the cycling of proline shuttles the NADPH generated from the shunt into a source of reducing potential for ATP generation. The source of the proline (and hydroxyproline) under conditions of nutrient stress is collagen in extracellular matrix. Collagen is the most abundant (by mass) protein in the body and 25% of the amino acid residues in collagen is either proline or hydroxyproline. Thus, degradation of collagen would provide proline and hydroxyproline as stress substrates. In published studies using the skin tumorigenesis model, dermal collagen is rapidly decreased. In tissue culture studies we have shown that the conditions which cause upregulation of POX are correlated with an increase in expression of matrix metalloproteinases (MMP2, MMP-9) and an increase in intracellular proline. The accumulation of proline in spite of an increased level of POX suggests that proline carbons are not totally oxidized. More likely, proline is cycling in a metabolic interlock with glucose metabolism in the pentose phosphate pathway so that the reducing potential (NADPH) generated by the pentose phosphate pathway can be shuttled into mitochondria as proline to generate ATP